A study of the interaction of Escherichia coli elongation factor-Tu with aminoacyl-tRNAs by partial digestion with cobra venom ribonuclease

A study of the interaction of Escherichia coli initiation factor IF2 with formylmethionyl‐tRNAMetf by partial digestion with cobra venom ribonuclease

“…Using the same technique, we have recently shown that EF-Tu:GTP protects the amino acid acceptor stem of different aminoacyl-tRNAs against cobra venom ribonuclease digestion [4]. In that case, we observed an increased intensity of the cuts in the anticodon stem when aminoacyl-tRNA bound EF-Tu:GTP.…”

“…ethanol after addition of 0.2Azbo units carrier tRNA. The precipitate was dissolved in 10 ~1 loading buffer 0.02 M Na-citrate (pH 5.0), 7 M urea, 0.001 M EDTA, 0.025% bromophenoi blue, 0.025% xylene cyanol) and analysed by high-voltage polyacrylamide gel electrophoresis [4]. In control experiments, IF-2 was omitted or GTP added to OS mM final con-38-32- …”

Conformational changes of aminoacyl‐tRNA and unchanged tRNA upon complex formation with polypeptide chain elongation factor Tu

“…GTP binds close to s2T(54) and stabilizes the tertiary structure around this residue. In fact, it has been shown that EF-Tu-GTP is in contact with the tandem of the aminoacyl-stem and the T-stem of aminoacyl-tRNA [5,6]. It should be noted that this conformational stabilization occurs regardless of the aminoacylation of tRNA~ I~.…”

“…Therefore, it is interesting to inquire why the complex of EF-Tu.GTP and aminoacyl-tRNA is much more effective in the Asite binding than other EF-Tu-tRNA complexes. Such differences in the binding activity are probably related to conformational differences among EF-Tu.tRNA complexes, because ribonuclease digestion experiments have suggested that the conformation of aminoacyltRNA changes upon binding with EF-Tu-GTP [5,6]. Therefore, it is important to compare the conformation changes of tRNAs (aminoacyl-tRNA and uncharged tRNA) upon binding with EF-Tu.GTP or EF- Abbreviations: CD, circular dichroism; EF-Ts, polypeptide chain elongation factor Ts; EF-Tu, polypeptide chain elongation factor Tu; Ile-tRNA, isoleucyl-tRNA~Je; IleRS, isoleucyl-tRNA synthetase; sZT, 2-thioribothymidine Tu.…”

Structural features in aminoacyl‐tRNAs required for recognition by elongation factor Tu

“…The molecular basis of aminoacyl-tRNA recognition by EF-Tu . GTP, has been further investigated in 'footprinting' experiments performed with yeast Phe-tRNAPhe [58,59], and with E. coli Phe-tRNAPhe [59,60], Glu-tRNAGiU [59] or MettRNAp' [59,60] using a variety of single-and double-strand specific ribonucleases (RNase Ti, Tz or A, and cobra venom ribonuclease). From these studies it emerges that in addition to the single-stranded 3 ' -CCA end bearing the aminoacyl moiety, the EF-Tu .GTP complex efficiently protects the acceptor and the T stems of aa-tRNAs.…”